1-ß-D-Arabinofuranosylcytosine (ara-C), an anticancer drug, has been isolated for the first time from a natural source, the mushroom Xerocomus nigromaculatus HONGO. Mushrooms are well known to produce unusual compounds with a wide variety of biological activities, and a number of metabolites showing antimicrobial and cytotoxic activities have been isolated from them, such as pleurotellol, alliacol A, strobilurin, saponaceolide and so on. During the course of our screening for biologically active metabolites from mushrooms (Basidiomycetes), a potent cytotoxin (IC50 value for P388: 0.004 μg/ml), tentatively named Xn-I, was isolated from Xerocomus (X) nigromaculatus HONGO (Japanese name: Kuroazaawatake). After structural studies, this compound was identified as 1-β-D-arabinofuranosylcytosine (ara-C) on the basis of physico-chemical and spectral evidence (Fig. 1). We wish to report herein the first isolation of this synthetic anticancer drug, ara-C, from a natural source.